Validation and application of a SEC-HPLC method for the determination of total protein in therapeutic immunoglobulins as an alternative to the European Pharmacopoeia methods.

Several analytical procedures are described in the European Pharmacopoeia (Ph. Eur.) to determine total protein content. However, the method for the determination of protein content in therapeutic immunoglobulins prescribed in the Ph. Eur. monographs is the Kjeldahl method. The Kjeldahl method is time-consuming and requires the use of large amounts of hazardous reagents, which also results in the production of a large amount of hazardous chemical waste. The purpose of this work was to validate an alternative chromatographic method that requires no hazardous reagents and saves time, using the same instrumental conditions specified in the Ph. Eur. for the human immunoglobulin size-exclusion high-performance liquid chromatography (SEC-HPLC) molecular-size distribution assay. The chromatographic separation was achieved with a TSKgel G3000SW (600 × 7.5 mm, 10 µm) column, using an isocratic elution, with detection at 280 nm wavelength. The mobile phase consisted of an aqueous solution of 0.03 M disodium hydrogen phosphate dehydrate, 0.01 M sodium dihydrogen phosphate monohydrate, 0.2 M sodium chloride and 1 mM sodium azide. The protein content of the test samples was determined referring to a standard with a known protein concentration (i.e. Human immunoglobulin (molecular size) Biological Reference Preparation). The method was validated evaluating the characteristics precision and trueness according to the ICH Q2 guideline, and the goodness of linear fit for the signal response was assessed (given for information only). In addition, the equivalence of methods was evaluated with two one-sided t-tests (TOST) analysis with the Kjeldahl method mentioned in Ph. Eur. monographs on therapeutic immunoglobulins, and with Bland-Altman analysis of SEC-HPLC and manufacturers' data (Kjeldahl and biuret methods). The uncertainty of measurement was also calculated in order to evaluate the accuracy and quality of the results, thus facilitating a reliable compliance/non-compliance decision. Based on the outcome, the method is proposed as a suitable and convenient alternative for the determination of protein content in human immunoglobulins.

A significantly lower potency observed for the 3rd WHO International Standard for Parvovirus B19V DNA with the cobas TaqScreen DPX test.

BACKGROUND: In the context of the Official Medicines Control Laboratories plasma pool testing for Parvovirus B19 DNA, we use the cobas TaqScreen DPX test. When we re-evaluated this method using the 3rd B19 DNA WHO IS at the final concentration of 4 log IU/mL, we observed a titre lower than expected, i.e. 3.79 log IU/mL. Therefore, we further investigated the accuracy of the DPX test. MATERIALS & METHODS: The following B19V DNA materials were tested by using both the DPX test and an in-house real-time PCR: The 1st, 2nd and 3rd WHO ISs for B19V DNA The Non WHO B19V DNA Reference Material for NAT The Biological Reference Preparation B19 virus DNA for NAT testing, batch 1 . RESULTS: The DPX test showed a good accuracy for all B19V DNA materials with the exception of the 3rd WHO IS for B19V DNA. In fact, an underestimation of about 38% was observed for all dilutions of this standard with respect to the nominal titre. With the B19V in-house real-time PCR, all four materials proved to be well calibrated against the 1(st) WHO IS for B19V DNA, used as external standard curve. CONCLUSION: In this study, we demonstrated that the DPX test underestimates the B19V DNA content of the 3rd WHO IS for B19V DNA and that this is not due to an incorrect potency assigned to the standard but, most probably, to a mismatch between the primers/probe and the sequence of the target region in the 3rd WHO IS for B19V DNA.

External quality assessment programmes for detection of HCV RNA, HIV RNA and HBV DNA in plasma: improved proficiency of the participants observed over a 2-year period.

BACKGROUND AND OBJECTIVES: Two External Quality Assessment Programmes (EQAPs) were run in 2008 and 2009 to evaluate the proficiency of blood centres in detecting, by nucleic acid amplification techniques (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: In the EQAP-2008, three customized panels were designed; each containing positive samples with a viral nominal concentration for the three viruses of about three times the 95% DL of the respective commercial NAT assay. In the EQAP-2009, the proficiency of the participants was evaluated with a single panel, independently on the NAT method used. RESULTS: While 84% (102/122) of the participants in the EQAP-2008 correctly identified the positive and negative samples of the panels, in the EQAP-2009 the percentage of proficient laboratories increased to 97% (118/122). Most importantly, in this 2-year experience, we observed a decrease in the number of pre-/postanalytical errors, from 14 in 2008 to two in 2009. CONCLUSIONS: The design of these two EQAPs allowed participants to assess the performance of the NAT methods applied in their routine screening of blood donations, not only with respect to analytical errors but also to human errors that, despite the high level of automation reached by NAT methods, can still occur.

A preliminary assessment of the occupational risk of acquiring Legionnaires' disease for people working in telephone manholes, a new workplace environment for Legionella growth.

BACKGROUND: Telephone manholes (TMs) are underground wells, used in Italy by the telecommunication companies to locate telephone networks. Following a fatal case of Legionnaires' disease (LD), acquired during working activity in a TM, we investigated whether Legionella was present in TMs and could be a risk for manhole workers (MWs). METHODS: Three hundred fifty-three environmental samples were collected from 100 TMs, and serum antibody titer against Legionella pneumophila and Legionella species non-pneumophila was determined from both MWs and control non-manhole workers. RESULTS: L pneumophila and Legionella species non-pneumophila were detected in 28% of water samples, in 8% of the biofilm, and in 6% of sediment matrices taken in TMs, in a concentration range of 10(2) to 10(4) colony-forming units/L. No Legionella was found in TM air samples. Although there was a statistically significant difference (P = .027) in antibody titer to L pneumophila serogroup 1 (Lp1) between MWs and non-manhole workers, a multivariate logistic regression analysis showed a significant association between antibody against Lp1 and both age group and the practice of aquatic sports. CONCLUSION: Although further investigations will be performed to quantify the risk of acquiring legionellosis, this preliminary study demonstrates for the first time the presence of Legionella, including human pathogenic species, in a working environment such as TM.

Training with computer-supported motor imagery in post-stroke rehabilitation.

Converging lines of evidence suggest that motor imagery (the mental simulation of a motor act within working memory) is associated with subliminal activation of the motor system. This observation has led to the hypothesis that cortical activation during motor imagery may affect the acquisition of specific motor skills and help the recovery of motor function. In this paper, we describe a clinical protocol in which we use interactive tools to stimulate motor imagery in hemiplegic stroke patients, thereby helping them to recover lost motor function. The protocol consists of an inpatient and an outpatient phase, combining physical and mental practice. In the inpatient phase, patients are trained in a laboratory setting, using a custom-made interactive workbench (VR Mirror). After discharge, patients use a portable device to guide mental and physical practice in a home setting. The proposed strategy is based on the hypotheses that: (a) combined physical and mental practice can make a cost-effective contribution to the rehabilitation of stroke patients, (b) effective mental practice is not possible without some form of support, from a therapist (as in our inpatient phase) or from technology (as in the outpatient phase), (c) the inclusion of an outpatient phase will allow the patient to practice more often than would otherwise be possible, therefore increasing the speed and/or effectiveness of learning, and (d) the use of interactive technology will reduce the patient's need for skilled support, therefore improving the cost-effectiveness of training.

The VEPSY UPDATED Project: clinical rationale and technical approach.

More than 10 years ago, Tart (1990) described virtual reality (VR) as a technological model of consciousness offering intriguing possibilities for developing diagnostic, inductive, psychotherapeutic, and training techniques that can extend and supplement current ones. To exploit and understand this potential is the overall goal of the "Telemedicine and Portable Virtual Environment in Clinical Psychology"--VEPSY UPDATED--a European Community-funded research project (IST-2000-25323, www.cybertherapy.info). Particularly, its specific goal is the development of different PC-based virtual reality modules to be used in clinical assessment and treatment of social phobia, panic disorders, male sexual disorders, obesity, and eating disorders. The paper describes the clinical and technical rationale behind the clinical applications developed by the project. Moreover, the paper focuses its analysis on the possible role of VR in clinical psychology and how it can be used for therapeutic change.

Avatars in clinical psychology: a framework for the clinical use of virtual humans.

Early applications of virtual reality (VR) technology in psychological assessment, treatment, and research have yielded promising results. In particular, an increasing number of studies analyze the unique features of the experience made by patients during their exposure to virtual environments. However, the majority of these studies explore how patients navigate in the virtual spaces and interact with virtual objects. Only a few of them investigate the features of inhabited virtual environments, where real people and autonomous virtual humans are able to interact and to cooperate. In particular, there is a lack of discussion of the role that such autonomous virtual humans could have in VR-aided psychotherapy. The main goal of this paper is to identify a framework for future research in this area. Three levels of analysis are identified. The purpose of the first two levels is the identification of the key "physical" features (e.g., appearance, structure) and "internal" characteristics (e.g., behavior, degree of autonomy, perceptual capabilities) needed by an effective simulation. The third level is concerned with the evaluation of the interaction characteristics required for a successful relationship between the patient and the virtual human.

The VEPSY UPDATED project: technical and clinical rationale.

The emergence of new shared media, such as the Internet and virtual reality are changing the ways in which people relate, communicate, and live. Health care, and in particular clinical psychology, is one of the areas that could be most dramatically reshaped by these new technologies. To exploit and understand this potential is the overall goal of the "Telemedicine and Portable Virtual Environment in Clinical Psychology"--VEPSY UPDATED--an European Community funded research project (IST-2000-25323, http://www.vepsy.com) whose specific goal is the development of different PC based virtual reality modules to be used in clinical assessment and treatment. In particular the developed modules have been using to address the following pathologies: anxiety disorders; male impotence and premature ejaculation; obesity, bulimia and binge-eating disorders. The chapter details the general technical and clinical characteristics of the developed modules.

The VEPSY updated project: virtual reality in clinical psychology.

Many of us grew up with the naive assumption that couches are the best used therapeutic tools in psychotherapy. But tools for psychotherapy are evolving in a much more complex environment than a designer's chaise lounge. In particular, virtual reality (VR) devices have the potential for appearing soon in many consulting rooms. The use of VR in medicine is not a novelty. Applications of virtual environments for health care have been developed in the following areas: surgical procedures (remote surgery or telepresence, augmented or enhanced surgery, and planning and simulation of procedures before surgery); preventive medicine and patient education; medical education and training; visualization of massive medical databases; and architectural design for health care facilities. However, there is a growing recognition that VR can play an important role in clinical psychology, too. To exploit and understand this potential is the main goal of the Telemedicine and Portable Virtual Environment in Clinical Psychology--VEPSY Updated--a European Community-funded research project (IST-2000-25323, http://www.vepsy.com). The project will provide innovative tools-telemedicine and portable-for the treatment of patients, clinical trials to verify their viability, and action plans for dissemination of its results to an extended audience-potential users and influential groups. The project will also develop different personal computer (PC)-based virtual reality modules to be used in clinical assessment and treatment. In particular, the developed modules will address the following pathologies: anxiety disorders; male impotence and premature ejaculation; and obesity, bulimia, and binge-eating disorders.

Study of pressure pain and cellular concentration of neurotransmitters related to nociception in episodic tension-type headache patients.

The pressure pain threshold of 30 patients affected by tension-type headache was assessed and the values compared with those of a group of 30 age-matched control subjects. In the patient group, pressure pain threshold values were related to the blood cell concentration of some neurotransmitters which are considered to be involved in the genesis and modulation of pain (beta-endorphin levels in peripheral blood mononuclear cells [PBMCs], substance P and serotonin concentrations in platelets). The pressure pain threshold was significantly lower in tension-type headache patients than in control subjects (P < 0.0006). Significantly lower levels of beta-endorphins in PBMCs and substance P in platelets, as well as significantly higher levels of serotonin in platelets were found in tension-type headache patients compared to the control subjects (P < 0.0001). A significant positive correlation was found between pressure pain threshold values and beta-endorphin levels in both control and patient groups (P < 0.0001). On the contrary, a statistically significant negative correlation was evident between pressure pain threshold values and substance P levels in platelets in both patients and control subjects (P < 0.01 and P < 0.001, respectively). In both groups, there was a negative correlation between beta-endorphins in PBMCs and substance P in platelets (patients P < 0.02, controls P < 0.001). The findings of altered beta-endorphin levels in blood mononuclear cells and substance P levels in platelets could be the peripheral biochemical reflection of the low pressure pain threshold values in tension-type headache patients, and support the hypothesis of an impairment of the antinociceptive systems in this form of headache.

[Vaginal metastasis from renal adenocarcinoma. A clinical case]. / Metastasi vaginale secondaria ad adenocarcinoma renale. Caso clinico.

We describe a case in which vaginal bleeding has represented the onset symptom of a renal clear cell adenocarcinoma. A 53-year-old patient presented with a bleeding polypoid neoformation of the vaginal forewall for which exeresis was immediately decided for diagnostic and curative purpose. An attentive histological examination was immediately addressed towards the renal origin. Afterwards the search and image staging (abdominal and pelvic ultrasound examination, urography, abdominal and cerebral TAC) has allowed us to confirm the presence of a tumor of the left kidney and audit that the secondary vaginal location to the moment was probably the only manifestation of the illness. Left nephrectomy with complementary associated lymphoadenectomy joined to the auxiliary therapy with progestin and interferone has allowed us to decidedly improve the subjective and general conditions of the patient.